Antimutagenic treatment of bacteria

ABSTRACT

This invention is concerned with a method of killing bacteria with a bactericide so as not to inherently produce bacteria strains resistant to the bactericide. In the method of this invention, the bactericide is utilized along with an effective amount of an antimutagenic agent such as spermine, spermidine, quinacrine hydrochloride, chlorpromazine, promethazine, levomepromazine, cyclobenzaprine, 3-chlorodibenzocycloheptene, protriptyline, amitriptyline, azepine hydrochloride and imipramine.

United States Patent Sevag [4 1 Sept. 5, 1972 [54] ANTIMUTAGENIC TREATMENT OF BACTERIA [56] References Cited [72] Inventor: G. Sevag, Newtown UNITED STATES PATENTS quare, a.

2,579,185 12/1951 Granatek ..260/239.1 [73] Assignee: The Trustees of the University of Pennsylvania Primary Examiner-Sam Rosen 22 Filed: Sept. 4, 1969 [21] Appl.No.: 871,019 57 ABSTRACT I Related US. Application Data This invention is concerned with a method of killing bacteria with a bactericide so as not to inherently [60] Dvlslon of S produce bacteria strains resistant to the bactericide. In abandoned whlch a 0 the method of this invention, the bactericide is utilized a 3 3 J a3't f S N g zgg along with an effective amount of antimutagenic an a con 0 agent such as spermine, spermldme, quinacrine March 1964 abandoned hydrochloride, chlorpromazine, promethazine,

levomepromazine, cyclobenzaprine, 3-chlorodibenzo- [52] us. Cl. ..424/1s1, 32 1122 2 7 43511222587, cycloheptene, protfiptyline, amitriptyline, mpine h (1 hi d d l l 51] Int. Cl. ..A6lk 21/00, A61k 27/00 y roe 'm'pramme 9 Claims, No Drawings ANTTMUTAGENHQ TREATNT F BACTERIA This application is a divisional application filed under Rule 147 of application Ser. No. 576,566 filed Sept. 1, 1969, now abandoned which in turn was partly a continuation-in-part of application Ser. No. 297,200, filed July 24, 1963 now abandoned; and a continuation-impart of application Ser. No. 355,480, filed March 27, 1964 now abandoned.

This invention relates to a method for preventing the development of mutant strains of bacteria, and to bactericidal compositions containing antimutagenic agents.

One of the major problems presented in attempting to control and eliminate developments of Staphylococcus aureus, Aerobacter aerogenes and Escherichia coli, for example, is the emergence of drug-resistant populations of such bacteria. When Staphylococcus aureus is treated with limited dosages of streptomycin, an antibiotic, mutation gradually occurs and resistant strains of Staphylococcus aureus develop, which strains become progressively more resistant to repeated treatments with streptomycin. Thus, unless a massive dosage of streptomycin is applied at the outset, which completely eliminates all bacteria at once, control of the bacteria by contact treatment with streptomycin soon becomes impossible. Similar problems are encountered in treating these bacteria with other bactericidal agents, such as ultra-violet radiations.

Even when massive drug dosages are utilized in hospitals upon discovery of an outbreak of Staphylococcus aureus, for example, the application of the drug does not usually suffice to eliminate the bacteria entirely, and subsequent mutatory growth which produces drug-resistant strains renders the problem of control an extremely serious one. Similar problems exist with Aerobacter aerogenes, Escherichia coli and with other bacteria.

Accordingly it is an object of this invention to provide a composition and method for controlling bacteria wherein the emergence of such drug-resistant strains is prevented. Other objects and advantages of this invention will become apparent in further detail hereinafter.

It has now been discovered that the development of mutant populations of bacteria from various strains of gram positive bacteria such as Staphylococcus aureus and gram-negative bacteria such as Aerobacter aerogenes, Escherichia coli and other bacteria is prevented by incorporating into the growth system an antimutagenic agent selected from the group consisting of spermine, spermidine, quinacrine hydrochloride, chlorpromazine, promethazine, levomepromazine, cyclobenzaprine, 3-chlorodibenzocycloheptene, protriptyline, amitriptyline, azepine hydrochloride and imipramine.

It is believed that the antimutagenic agent cooperates to reduce bacteria to a common status, or common denominator, thereby preventing mutation. To demonstrate this, a series of tests was run. These tests have led me to believe that the antimutagenic agents of this invention combine with the nucleic acids which are the heredity factors in the bacteria, and that these then become resistant to change. In this manner, the tendency to produce mutant strains is eliminated.

These tests are exemplified by Example 1, as follows:

EXAMPLE l Various solutions in distilled water contained in dialysis bags were prepared (in duplicate) as follows:

distilled 500 g/ml each.

Each system in individual'bags was suspended in 20 ml. of distilled water as outside fluid, and each system was housed within an individual stoppered bottle to prevent evaporation. Aliquots were taken out at l, 2, 3, 4, 5 and 22 hour periods and analyzed for the spermine content using the colorimetric ninhydrin method. Systems 1 and 2 remained clear and complete dialysis of spermine occurred. System 3 was observably turbid, and quantitative analysis showed that 950 g of spermine had complexed with DNA. It will be appreciated that when mutant strains are developed in the absence of an antimutagenic agent, some of the mutant strains are more resistant and some are less resistant to the repeated action of a bactericidal agent, but the resistant strains nevertheless survive. Thus, if mutant-resistant strains are already in existence, the application of spermine in conjunction with the bactericidal agent cannot convert the resistant strains back to non-resistant strains, but if no resistant strains are initially present, spermine or spermidine in combination with the bactericidal agent effectively prevents the development of resistant strains and therefore renders the bactericidal agent fully effective. This property is particularly valuable in hospitals and in other areas where resistant strains have a tendency to develop. Where strains have developed which are resistant to only one drug or other bactericidal agent, for example streptomycin, then these resistant strains can nevertheless be treated successfully with another such agent, i.e. penicillin combined with an antimutagenic agent.

It has also been found that since a bacterial strain which has become resistant to one particular bactericidal agent has a pronounced tendency to become resistant to others causing a real danger that a resistant strain will be developed during testing with a second or third antibiotic, this invention has special utility in laboratory pre-testing of bacteria to determine which antibiotics will be effective treating agents. The danger of emergence of strains resistant to more than one bactericidal agent is eliminated by incorporating spermine or spermidine with the antibiotics used in the test to which the organism is sensitive. Otherwise, even with a massive dosage of a bactericidal agent, resistant bacteria may develop.

While Staphylococcus aureus and Aerobacter aerogenes or Escherichia coli have been referred to specifically herein, this invention is not restricted to the control of these organisms but applies broadly to the gram-positive and gram-negative bacteria of which these are representative.

In accordance with an embodiment of this invention, an organism such as Staphylococcus aureus or Aerobacter aerogenes, cultured naturally or in a synthetic medium, is treated with a sterile solution of any one of the specific bactericidal agents heretofore referred to, such solutions preferably being in distilled water. Spermine or spermine phosphate (or spermidine or spermidine phosphate) solutions are sterilized by filtration through .Millipore disc filters. Repeated applications of such solutions, coadministered with the antibiotic in accordance with this invention, result in a complete elimination of the bacteria. Streptomycinspermine solutions are preferably administered by injection, while penicillin-spermine solutions may be administered by injection or orally. The methods of administering these and other antibiotics are determined by the preferred method of administering the antibiotic along.

In accordance with this invention, sterile solutions of the antibiotics and distilled water are used, and the spermine or spermidine may be incorporated into this solution either as such or in any other desired form such as the phosphates, in an amount of about -100 micrograms per milliliter.

Sperrnine has the formula li NCl-l (CH l-INCl-l (CH NH(CH CH NH Spermidine is a shorter chain polyamine and has the following formula:

H NCH (CHC2) NH(CH CH NH These compounds may be utilized per se or as the phosphates, in which form they occur naturally.

EXAMPLE2 A growth medium was prepared consisting of Staphylococcus aureus (3A) and this was cultured in a synthetic growth medium containing 16 amino acids, vitamins and salts. The growth medium was in accordance with the disclosure of Steers and Sevag, Arch Biochem. 24, 129 (1949). Two H. Staph. aureus strains were grown in extract broth containing 0.5 percent glucose, and A. aerogenes in a minimal salts-glucose medium according to the method of Drabble and Hinshelwood, Sir Cyril Roy. Soc. (B) 154, 449 (1961).

An inoculum containing from 1 to 2 X 10 cells was added to 5 ml. of the appropriate liquid medium in a 14 X 120 mm. test tube. For the staphylococcal strains the inoculated cells were taken from the second daily transplant on extract agar from the stock culture. These cells were suspended in a 1/15 molar phosphate buffer at a pH of 7.34. Cells of A. aeragenes taken from the stock culture were grown twice in salts-glucose medium and washed in a phosphate buffer solution having a pH of 7.1 before use.

All cultures were incubated at 37 C. Growth was followed by measuring the increase in turbidity using a Klett-Summerson photoelectric colorimeter with filter No. 56. The instrument was calibrated against viable plate count and microscopic count for each strain. Sterile solutions of the antibiotics in distilled water were used. Spermine or spermine phosphate solutions were sterilized by filtration through Millipore disc filters. For colony counts, pour plates were prepared from the synthetic amino acid medium supplemented with 1.5 percent agar were incubated at 37 C.

The results with respect to various strains of S. aureus (3A) in media containing spermine, streptomycin and spermine plus streptomycin appear in the following table.

TABLEl Prevention of the Emergence of Drug-Resistant Cells from the Drug-Sensitive Population of Staphylococcus aureus (3A) in the Combined Presence of Sperrnine and Streptomycin, and Their Failure to Affect the Growth of the Streptomycin-resistant Strain Growth Drug- Sensitive Drug-Sensitive Drug-Sensitive System Normal Strain strain after 6 strain* after subcultures in one subculture Spermine in Streptomycin micrograms/ml 100 micrograms/ Growth Turbidity Readings at Hours of:

Additions 0 528 36 46 101I) l8 42 1000 8 27 47 grams/ml 15 *Similarly, the combined presence of spermine and streptomycin had no effect on the growth of the normal strain after 4 sub-cultures in streptomycin followed by 6 subcultures in spermine.

From the foregoing table it will be apparent that spermine alone has little or no delaying action on the growth of these three strains. In streptomycin, the resistant strain grows readily and even the sensitive strains produce resistant cultures within 46 hours. The growth of cells which are initially fully resistant is not affected by the combined presence of spermine and streptomycin, just one subculture in streptomycin being sufficient to render the cells resistant to this combination. On the other hand, cultures of the sensitive strain are prevented from developing into a resistant population when spermine is present. Even sperminegrown cells are inhibited by spermine and streptomycin administered together.

Tests with A. aeragenes yielded. results similar to those with S. aureus (3A).

The foregoing results show that spermine itself does not exercise a bactericidal action. If it had exerted a bactericidal action the viable count of a sensitive inoculant could be sufficiently reduced to deplete the population of resistant mutants. Viable colony counts, however, showed that S. aureus growing in 10 micrograms of spermine per ml. alone were not killed but multiplied 26-fold in a 9 hour growth period. When growing in 100 micrograms of spermine per ml. there was first a 9 hour lag with no increase or reduction in viable count, followed by a 30-fold increase during the second 9 hours. This confirms the fact that spermine itself does not exercise a bactericidal action.

EXAMPLE 3 A mixture of a small number of cells of A. aerogenes resistant to streptomycin (2 cells) and 0.57 X 10 sensitive cells was used as the inoculum. This readily yielded a resistant population in the combined presence of spermine and streptomycin. However, sensitive cells alone, with no resistant cells added, failed to develop in this medium, as appears in the following table.

TABLE 2 Failure of the Emergence of Streptomycin-resistant Bacteria from Sensitive lnoculum and the Ready Emergence of Resistant Bacterial Population when only 2 Resistant Cells were added to the Inoculum Containing 0.57 X Sensitive Cells/5 m1. Medium The foregoing illustration confirms the fact that cultures of the sensitive strain do not contain any of the resistant culture, since otherwise such resistant cells would selectively multiply and yield a drug-resistant population even in the combined presence of spermine and the antibiotic.

EXAMPLE 4 The following table shows the results obtained in testing the penicillin-sensitive hospital strain of Staphylococcus aureus.

TABLE 3 Prevention of the Emergence of Drug-resistant Cells from Penicillin-sensitive and Penicillin-resistant Strains of Staphylococcus aureus in the Combined Presence of Spermine and Penicillin or Streptomycin Growth System Staphy- Growth Turbidity Readings lococcal at Hours of: Strains Additions 0 21 44 68 91 163 None Sensitive 24 61 161232 Spermine (100 micro to Peni- -grams/ml.) cillin 24 54 70 90 105 132 Penicillin 0.1 1357) unit/ml.) 24 46 89 112110 123 Penicillin (0.1 unitlml.)+spermine (100 micrograms/m1.) 27 31 31 31 30 33 None 21 71 218 Spermine (100 micro Resistant -grams/ml.) to Peni- 19 54 144 200 Penicillin (1 unit/ cillin ml.) #1360) 23 63 265 Penicillin (1 unit/ ml.) Spermine (100 micrograms/ml.) 21 40 137232 None Resistant 25 105 310 Spermine (100 micro to Peni- -grams/ml.) cillin 25 72 162276 Streptomycin (50 1360) micrograms/ml.) 24 25 32 93 310 Streptomycin (50 micrograms/ml.) Spermine (100 micro -grams/rnl.) 22 26 28 35 30 31 micrograms of streptomycin per ml. in combination with spermine.

Many tests were run to deten'nine the minimal effective doses for Staphylococcus aureus (3A). These showed that 10 micrograms of spermine per ml.

together with 100 micrograms of streptomycin per m1. prevented the emergence of resistant cells. 50 micrograms of streptomycin with 100 micrograms of spermine per ml. produced the same effect on the penicillin-resistant hospital strain of Staphylococcus aureus. Obviously, the quantities of spermine and spermidine and phosphates thereof will be found to vary beyond the range of 10-100 micrograms per ml. depending upon the bacteria involved, but the polyamine must be present in any event in an amount sufficient to eliminate and to prevent emergence of resistant cells.

' Similar results are obtained when bactericidal means other than antibiotics are utilized for killing the bacteria. For example, ultra-violet irradiation of cultures, utilized in conjunction with cells which were pre-incubated with 100 micrograms of spermine per ml. for 40 minutes, produced a viable count of cultures which was approximately 170 times less than the cultures similarly treated with ultra-violet light, spermine having been omitted. Accordingly, it will be appreciated that the essence of the invention resides in treating the bacterial medium with spermine prior to the time that the medium is affected by the killing agent which may be of the aforementioned antibiotics or even ultra-violet light as well.

In view of the foregoing disclosure, it will be appreciated that spermine or spermidine in combination with streptomycin, penicillin, erythromycin, tetracycline of chlorampenicol, or utilized in conjunction with ultra-violet light, can completely prevent the emergence from sensitive cells of populations which are resistant to ghe drugs or to the ultra-violet light. This effect is not comparable to the usual synergism between two drugs acting together, since spermine or spermidine alone fails to have any bactericidal action on the strains tested and small numbers of resistant cells can survive in a medium containing both spermine and antibiotic. Spermine is believed to intervene during development of the resistance mechanism, but cells which are already fully resistant are unaffected by the presence of the spermine.

In accordance with an embodiments of this invention, an organism such asv Staphylococcus aureus or Eschericia coli, cultured naturally or in a synthetic medium, is treated with a sterile solution of any one of the specific bactericidal agents heretofore referred to, in combination with quinacrine hydrochloride, such solutions preferably being in distilled water. Repeated applications of such solutions, coadministered in accordance with this invention, results in a complete elimination of the bacteria. Streptomycin-quinacrine hydrochloride solutions are preferably administered by injection, while penicillin-quinacrine hydrochloride solutions may be administered by injection or orally.

Quinacrine hydrochloride has the empirical formula C l-I Cl N O-2H Cl-2H O and a molecular weight of 508.91 Its structural formula can be characterized as:

The pharmacology of quinacrine hydrochloride is well. known. It is widely used clinically in the treatment of malaria and is administered in dosages as high as one gram per day without adverse effects. The dosage effective for administration to human patients according to this invention in combination with bactericidal agents will generally be considerably less than 1 gram per day.

EXAMPLE 5 A culture of Escherichia coli (strain B) prepared in a synthetic growth medium, 2 X cells per 5 milliliter of salts-glucose casein hydrolyzate, and incubated at 37 C was tested. The growth progress of the culture was determined by measuring the increase in the turbidity of the medium using a Klett-Summerson photoelectric colorimeter equipped with a number 56 filter. The instrument was calibrated against viable plate count and microscopic count for each strain. Sterile solutions of the antibiotics in distilled water were used. Solutions of quinacrine hydrochloride were sterilized by filtration through Millipore disc filters. For colony counts, pour plates were prepared from the synthetic medium supplemented with 1.5 percent agar and incubated at 37 C.

TABLE 4 Prevention of the Emergence of Drug-Resistant Cells from the Normal Strain of Escherichia coli (Strain B) in the Combined Presence of Streptomycin and Quinacrine Hydrochloride.

2 X 10 cells/5 ml of salts glucose casein hydrolyzate medium.

inoculum:

The results presented in Table 4 show that Escherichia coli (strain B) capable of yielding resistant strains in the presence of 10, or 30 micrograms per milliliter of streptomycin and unaffected by 40 micrograms per milliliter of quinacrine hydrochloride alone, showed a remarkable resistance to the growth of such strains in the presence of small concentrations of streptomycin (10 micrograms per milliliter) and quinacrine hydrochloride (40 micrograms per milliliter) together. It should be appreciated that the figures in Table 4 for the combination of quinacrine hydrochloride and streptomycin represent no growth whatever as confirmed by pour plate analysis. The medium acquires a natural darkening with age independent of the turbidity measurements and the colorimeter was not re-zeroed for this darkening phenomenon. The results are highly significant when it is appreciated that the synthetic medium is capable of producing growth of the observed very high order of magnitude. It should also be noted that the quinacrine hydrochloride by itself exerts no bactericidal action. When the growth turbidity readings at 28 hours are compared for inoculum in the growth medium without additions and for inoculum in the growth medium with 40 micrograms per milliliter of quinacrine hydrochloride added, the growth in both cases reached the same order of magnitude.

EXAMPLE 6 Table 5 demonstrates that the combination of micrograms per milliliter of streptomycin and 50 micrograms per milliliter of quinacrine hydrochloride prevented the emergence of a staphylococcal strain capable of developing resistance to streptomycin alone and unaffected by treatment with quinacrine hydrochloride alone.

TABLE 5 Prevention of the Emergence of Drug-Resistant Cells from the Normal Strain of Staphylococcus aureus (strain 3A) in the Combined Presence of Streptomycin and Quinacrine Hydrochloride.

inoculum:

1.0 X 10 cells/5 ml of 0.5% glucose nutrient broth. The washed inoculum was harvested from an overnight culture grown in the same medium.

Growth System Additions numbers in micrograms Growth Turbidity Readings at Hours of:

Reading 118 at 187 hours.

EXAMPLE 7 Table 6 demonstrates that staphylococcal strains which have already become resistant to streptomycin can be destroyed by the combination of 50 micrograms per milliliter of sulfathiazole and 100 micrograms per milliliter of quinacrine hydrochloride.

TABLE 6 Prevention of the Emergence of Sulfathiazole-Resistant Staphylococci by quinacrine hydrochloride.

lnoculum:

ca 50 cells/5 ml of salts-glucose case in hydrolyzate medium.

Strain resistant to 1000 micrograms per millileter of Streptomycin None 1 Sulfathiazole, 1 1

Sulfathiazole, 3

Quinacrine Hydrochloride, 0

Sulfathiazole, 50

and Quinacrine Hydrochloride,

Sulfathiazoie, 100

and Quinacrine Hydrochloride, 8 9

EXAMPLE 8 Table 7 demonstrates that Escherichia colistrains which have already become resistant to streptomycin can be destroyed by the combination of 10 micrograms per milliliter of sulfathiazole and 50 micrograms per milliliter of quinacrine hydrochloride.

TABLE 7 Prevention of the Emergence of Sulfathiazole-Resistant strains of Escherichia coli by Quinacrine Hydrochloride.

lnoculum:

ca 1 X 10*ce11s/5 ml of salts-glucose-casein hydrolyzate medium.

Growth System Sensitive strain Strain resistant to Additions num- (B) 1000 micrograms per bars in micromilliliter of Strepgrams per tomycin milliliter Growth turbidity readings at hours of:

0 18 4-2 71 137 160 0 18 4-2 71 137 160 None 10 161 197 4- 160 144 Sulfathiazole, 5 1 85 140 4- 160 124 Sulfathiazole, 5 6 16 98 300 13 172 Sulfathiuzo1e,l0 4- 8 7 6 134 157 8 12 15 112 222 Quinacrine Hydrochloride, 50 9 113 174 8 105 127 Quinacrine Hydrochloride, 100 61 184- 0 0 17 B7 154 Sulfathiazole, 5

and Quinacrine Hydrochloride.

50 Sulfathiazule, 10

and Quinacrine -r'ydrochloride,

O Sulfathiazole, l

and Quinacrine Hydrochloride,

1 Sulfathiszole. 5

and Quinacrine Hydrochloride,

Sulfathiazole, 10

and Quinacrine Hydrochloride.

EXAMPLE 9 Table 8 demonstrates the effectiveness of various bactericidal agents in combination with quinacrine hydrochloride in the staphylococci. of the emergence of resistant strains of staphlococci.

TABLE 8 Prevention of the Emergence of Novobiocin, Erythromycin and Tetracycline-Resistant Strains of Staphylococci by Quinacrine Hydrochloride.

lnoculum:

ca 1 X 10 cells/5 ml salts-glucose-cascin hydrolyzate medium.

Growth System Additions Growth Turbidity Readings numbers in micrograms at Hours of: permilliliter 0 22 46 118 148 168 None 8 282 285 280 Quinacrine Hydrochloride, 50 3 225 220 195 Quinacrine Hydrochloride, 100 8 216 220 215 Novobiocin,5 7 14 20 214 Novobiocin, 5 and Quinacrine Hydrochloride, 50 7 13 20 45 162 173 Novobiocin, 5 and Quinacrine Hydroch1oride,l00 9 18 28 27 25 24 Erythromycin,2.5 12 20 225 Erythromycin,5.0 2 16 13 226 Erythromycin,2.5 and Quinacrine 3 19 19 27 202 Hydrochloride, 50 Erythromycin, 5.0 and Quinacrine Hydrochloride, 50 2 ll 13 17 16 20 Erythromycin, 2.5 and Quinacrine Hydroch1oride,100 2 9 10 17 20 22 Erythromycin, 5.0 and Quinacrine Hydroch1oride,100 5 20 30 30 32 30 Growth System Additions Growth Turbidity Readings numbers in micrograms at Hours of: per milliliter 0 18 43 65 145 209 Tetracyc1ine,1 5 18 16 16 235 Tetracycline, 2.5 3 16 18 18 21 250 Tetracycline, 1 and Quinacrine Hydrochloride, 0 13 8 8 18 193 Tetracycline, 2.5 and Quinacrine. Hydrochloride, 80 0 12 14 16 25 33 Tetracycline, 1 and Quinacrine Hydroch1oride,160 2 16 20 17 20 27 Tetracycline, 2.5 and Quinacrine Hydroch1oride,l60 3 15 21 20 26 30 In accordance with an embodiment of my invention, it has been found that the phenothiazine tranquilizers, such as chlorpromazine, promethazine and levomeprazine, and the antidepressants, such as 3- chloro-dibenzocycloheptene, cyclobenzaprine, protriptyline, amitriptyline, desipramine and imipramine are effective in preventing the emergence of resistant strains of Staphylococcus aureus and Escherichia coli in the presence of bactericidal agents. By comparison, the antimalarials, such as chloroquine and hydroxychloroquine were found to be relatively ineffective in preventing such emergence. Moreover, the active compounds by themselves do not exercise a bactericidal action at what wouldnormally amount to effective concentrations, and as stated heretofore, the action is not synergistic.

The nomenclature and formulas of these antirnutagenic compositions follows:

TABLE 9 Chloroquine Diphosphate; 7-Chloro-4- (diethylarninol -methylbutylamino)quinoline diphosphate.

l monomomonm a. .2? V. V.

CHzCHzOBI E EEFK a Hydroxychloroquine; 7-chloro-4-[4-ethyl(2-hydroxyethyl )amino] l -methylbutylamino )quinoline.

Quinacrine Hydrochloride; 3-Chloro-7-methoxy-9- l-methyl-4-diethylaminobutylamino )acridinedihydrochloride.

Promethagine; l 0- Dimethylaminopropyl)phrenothiazine.

( JHa Levomepromazine; 2-methoxyl O-( 22 dimethylaminopropyl)phenothazine.

-methyl-3- Desiprarnine; 5-( 3-methylaminopropyl 1 0, l l dihydro-5l-l-dibenz-(b,f)azepine hydrochloride.

Cyclobenzaprine; N,N-Dimethyl-5H-dibenzo[a,d]-

cycloheptene-AS,-propylamine hydrochloride.

3-Chloro- 10,1 l-dihydro-N,N-dimethyl-5 l-ldibenzo[a,d]-cycloheptene-5-proplyamine hydrochloride.

\N/ or JHzCH2 HrN 11a);

Chlorpromazine; 2-Chloro-l0-( 3- dimethylaminopropyl)-phenothiazir1e.

H CHzCHzCI-IzNHCHz-IICl 13 .Protriptyline; N-Methyl-l-l-dibenzo[a,d]-cycloheptene-5-propy1amine hydrochloride.

V CHzCHrN(CH3)2-HC1 Amitriptyline;

dibenzo-[a,d] l ,4]-cycloheptadiene hydrochloride.

Each of the compounds identified in Table 9 was paired with either streptomycin, chloromycetin, or sulfathiazole, and the combined chemotherapeutic agents were tested for their effectiveness in preventing the emergence of drug-resistant strains of two organisms, i.e., Staphylococcus aureus and Escherichia coli. The specific organisms used in the following examples 5-(3-Dimethylaminopropylidene)- 14 Escherichia coli (B). Nutrient broth containing 0.5 percent glucose was used for overnight cultures. For making the analyses shown in the examples, the salts-glucose-casein-hydrolyzate medium was used. One liter of the medium contained 10 g. casein hydrolyzate, 10 g. phosphate, 1.7 g. NaCl, 0.07 g. MgSO -7H O, 0.19 g. (NH )PO 5 g. glucose, mg. tryptophane, 1 mg. nicotinamide, 1 mg. thiamine. For colony count, plating was carried out on nutrient agar. For the preparation of inoculum it is necessary to have a fresh slant to be used for the innoculation of the overnight glucosebroth cultures. This is centrifuged, washed with and suspended in M/ phosphate buffer of pH 7.35 and diluted appropriately to obtain the desired inoculum according to the standard curve. The solutions of antibiotics are sterilized by passing through a sterilizing filter. Colony counts were made in accordance with standard procedures. All test systems containing the phenothiazine-type tranquilizers were shielded by aluwere Staphylococcus aureus (3528, ATCC) and 20 minum foil against light.

PREVENTION OF THE EMERGENCE OF DRUG-RESISTANT STRAINS FROM THE SENSITIVE POPULATION OF STAPHYLOCOCC'USAURE US (3528) BY TRANQUILlZERS 1N COMBINATION WITH A DRUG Growth turbidity readings at hours oi- Terminal test for surviving Growth system (additions rig/ml.) 0 20 42 115 130 163-234 cells* 1. Control 4 255 276 294 2. Sulfathiazole, 50-100.. 21 238 c 3. Streptomycin, 30-50.. 0 238 4. Chloromycetin, 5-10. 7 5. Chloropromazine, 30. l 205 0. Promethazine, 100.. 5 160 7. Levorneprazine 5 8. Suliathiazole, 100 plus chlorpromazine, 30. 14 17 None. 0. Suliathiazole, 50 plus promethazine, 100. 8 21 Do. 10. Suliathiazole, 100 plus levomeprazinc, 100 5 7 Do. 11. Streptomycin, 30 plus chlorpromazine 30- 2 3 Do. 12. Streptomycin, 30 plus promethazine, 75.. 1 9 13. Streptomycin, 50 plus levomcprazine, 100 4 6 14. Chloromyeetin, 10 plus chlorpromazine, 30. 11 15 D 15. Chloromycetin, 5 plus promethazine, 75. 4 12 Do. 16. Chloromycetin, 5 plus levomeprazine, 100. 15 17 Do. 17. Chloromycetin, 75 plus levomeprazine, 80. 5 8 Do.

*0.2 ml. of the growth system spread out on nutrient agar plate.

N orE.-Medium; Salts-glucose-cascin hydrolysate medium with 1% phosphate p11 7.35. Inoculum; 1X10 cells/5 ml. medium containing suliathiazole and 1x10 cells/5 ml. medium containing streptomycin or chloromycetin.

EXAMPLE 1 1 ant to 1,000 g. of streptomycin/ml.

Growth turbidity readings at hours 01-- Growth system (additions ig/ml.) U 67 91 163 1. Control 2 220 2. Streptomycin, 1,000 4 226 3. Chlorpromazine, 30 7 190 180 4. Streptomycin 1,000 plus chlorpromazine, so 5 00 105 5. Promotnazlne 75, 100 4 142 150 0. Streptomycin, 1,000 plus promethazine,

100 3 18 85 7. Levomeprazine, 120 3 23 55 08 150 8. Streptomycin, 1,000 plus lcvomeprazine, 120 3 5 12 -10 77 185 EXAMPLE 12 Prevention of the emergence of drug-resistant strains of Staphylococcus aureus (3528) by certain antidepressants Growth turbidity readings at hours of- Surviving Growth systems* (additions poJml.) 0 18 42 67 90 114 138 210 cells Control 18 100 258 2. Cyclobenzaprine, 75 10 22 30 3. 3-chloro-cyeloheptene, 20, 30 5 31 39 4. Protriptyline, 50 5 23 72 5. Amitriptyline, 75. 5 23 35 0. Imipramine, 100 10 30 85 7. Azepine hydrochloride" 10 26 85 8. Streptomycin, 30 .v 6 14 ll. Streptomycin, 30 plus cyclobenzaprine, 75 3 3 5 10. Streptomycin, 30 plus 3-chlorooycloheptene, 30. 12 15 15 11. Streptomycin, 30 plus protrlptyllne, 50 7 I 7 8 12. Streptomycin, 30 plus amitriptyline, 75... 10 10 12 13. Streptomycin, 30 plus imipramine, 100 4 0 7 14. Streptomycin, 30 plus azepine hydrochloride, 75"*.. 8 7 7 EXAMPLE l2-Continued Prevention of the emergence of drug-resistant strains of Staphylococcus aurcus (3528) by certain antidepressants Growth turbidity readings at hours of- Snrvivin Y Growth systems (additions [ML/m1.) 18 42 67 90 114 138 211) cells l5. Suliathiazole, 50 .1 6 204 16. Sulfathiazole, 60 plus cyclobenzaprine, 75 0 2 2 Ho. 17. Sultathiazole, 50 plus 3-chloro-cyeloheptene, 20. 2 2 2 Do. 18. Sultathiazole, 50 plus protriptyline, 50.- U 0 0 Do. 19. Sulfathiazole, 50 plus amitriptyline, 75 O 2 2 Do, 20. Sulfathiazole, 50 plus imipramine, 100 13 Y 13 13 D0. 21. Suliathiazole, 50 plus azepine hydrochloride"* 14 14 r 14 15 Do.

"Growth medium consisted of 1% casein hydrolyzate, 0.5% glucose, 1% phosphate pli 7.35 and salts. Inocnlum. 1.0)(10 cells/ ml. in streptomycin and 1.0)( cells/5 ml. in sulfathiazole growth systems.

" l he presence or absence of surviving cells was determined by inoculating 0.2 ml. of test systems into 5 ml. of glucose broth and 0.2 ml. onto the nutrient agar plate.

"'Azeplne hydrochloride is 10,1l-dihy(lro-5-(ii-methyaminopropy EXAMPLE l3 l)-5H-dibenz(b,l) uzepine hydrochloride.

Prevention of the emergence of drug-resistant strains of Escherccllia coli by certain antidepressants Growth turbidity reading at hours (if-- Surviving Growth systems" (additions gJml.) 0 19 42 66 92 115 139 186 cells 1. Control 0 445 2. Sulfathiazole, 2. 3 r 3. Sultathiazole, 4 o 4. S-chloro-dibenzocycloheptcne, 50, 75.. 7 5. Cyclobenzaprine, 75 5 6. Protriptyline, 50 8 7. Amitriptyline, 75, 100 0 8. Azepine hydrochloride, 5() (i U. Azepine hydrochloride, 75 4 10. Imipramine, 75, 100 0 11. Sulfathiazole, 4 plus atabrine, 100 6 None 12. Sulfath'iazole, 2 plus 3-chloro dibenzoeycloheptcno, 75 0 0 2 4 4 Do. 13. Sulfothiazole, t plus cyclobenzaprine, 75 (l 0 0 U 1 Do. 14. Sultathiazole, 4 plus protriptyline, 50- 17 17 18 18 18 Do. 15. Sulfathiazole, 2 plus amitriptyline, 100"... 12 12 13 13 13 Do. 16. Sullathiazole, 2 plus azepine hydrochloride, 75. 0 a 0 1 Do. 17. Sultathiazole, 4 plus imipramine, 75 0 0 2 18. Streptomycin, 7.5 plus atabrine 150 7 9 12 18 Do. 19. Streptomycin, 7.5 plus 3-chlorodibenz0cyclohep- 6 (l 8 10 10 D0.

tene, 50. 20. Streptomycin, 7.5 plus cyclobenzaprine, 75 5 5 5 5 Do. 21. Streptomycin, 5 plus protriptyline, 50. 8 10 10 10 10 Do. 22. Streptomycin, 7.5 plus amitriptyline, 75 10 10 10 10 10 Do. 23. Streptomycin, 5 plus azepine hydrochloride, 75. 5 7 7 7 7 Do. 24. Streptomycin, 5 plus imipromine 100..." 12 12 12 13 16 Do.

*Inoculurn, l l0 cells/51111. salts-glucose casein hydrolyzute medium containing 1% phosphate of p11 7.35.

EXAMPLE 14 Growth of S. aureus (3628) by plate count in the presence of 3-chlorodibcnzocyclopheteno omitriptylino or azepine hydrochloride Cells/ml. at hours of System g/ml.) 0 l 3 5 7 24 49 (1) Control 1.39X10 1. 36 10 231x10 4. 05 10 9.1X10 9. 65x10 2. 9X10 (2) 3-chloro.* compound, 1. 23 10 1. 1X10 0. 71 10 1. 29X 10 1. 14x10 2. 3X10 6. x10 (3) Am1t r1ptyllne, 75 1. 32x10 0. X10 1. 07x10 1. 47x10 1. 78 10 2. 8X10 1. 62x10 (4) Azcplne hydrochloride, 75 1. 2x10 0. 7X10 1. 1X10 0. 97x10 1. 34 10 4. 8x10 1. 78x10 *3-chlorocycloheptene, 30.

Examples 10, 12 and 13 show that certain phenothiazine tranquilizers, such as chlorpromazine,

promethazine, or levomepromazine, the antidepressant arnitriptyline and its derivatives, imipramine, and its derivative azepine hydrochloride, when used in combination with a bactericidal agent selected from the classconsisting of sulfathiazole, streptomycin, and

Nora-Medium, Salts'glucose-casein hydrolyzate with 1% phosphate of pH 7.35.

anti-malarial chloroquines, derivatives of quinoline, in identical test systems are ineffective to prevent the emergence of Escherichia coli and Staphylococcus aureus strains resistant to all three drugs used individually. Furthermore, chloroquine in as high a concentration as 2,500 mg./ml. does not inhibit growth and failed to prevent the emergence of Escherichia coli strains resistant to streptomycin. On the other hand, as herein described, certain antidepressants and certain tranquilizers prevented the emergence of the strains of both bacterial species resistant to the drugs used.

Having thus described may invention, I claim:

1. In a method of killing bacteria by the action of a bactericidal agent selected from the class consisting of sulfathiazole, chloromycctin, streptomycin, penicillin, erythromycin, tetracycline, chloramphenicol and novobiocin, which bacteria normally develop strains resistant to the activity of said bactericidal agent, the step which comprises physically contacting bacteria which has not developed a resistant strain to said bactericidal agent with (1) said bactericidal agent and (2) an amount of the antimutagenic agent quincrine hydrochloride effective to prevent said bacteria from developing a resistant strain to said bactericidal agent.

2. A composition for killing bacteria by physical contact therewith consisting essentially of 1) an effective amount of a bactericidal component selected from the class consisting of sulfathiazole, chloromycetin, streptomycin, penicillin, erythromycin, tetracycline, chloramphenicol and novobiocin, and (2). an amount in the range of 20-200 micrograms of quinacrine hydrochloride, in sterile aqueous solution.

3. A composition for killing bacteria by physical contact, comprising a sterile aqueous solution of about -100 micrograms per milliliter or streptomycin and about -200 micrograms per milliliter of quinacrine hydrochloride.

4. A composition for killing bacteria by physical contact, comprising a sterile aqueous solution of about 0.0l-l.0 unit of penicillin per milliliter and about 20-200 micrograms per milliliter of quinacrine hydrochloride.

5. A composition for killing bacteria by physical contact comprising a sterile aqueous solution of about 0.1-10 micrograms per milliliter of novobiocin and about 20-200 micrograms per milliliter of quinacrine hydrochloride.

6. A composition for killing bacteria by physical contact comprising a sterile aqueous solution of about 0. l-10.0 micrograms per milliliter of erythromycin and about 20-200 micrograms per milliliter of quinacrine hydrochloride.

7. A composition for killing bacteria by physical con- 

2. A composition for killing bacteria by physical contact therewith consisting essentially of (1) an effective amount of a bactericidal component selected from the class consisting of sulfathiazole, chloromycetin, streptomycin, penicillin, erythromycin, tetracycline, chloramphenicol and novobiocin, and (2) an amount in the range of 20- 200 micrograms of quinacrine hydrochloride, in sterile aqueous solution.
 3. A composition for killing bacteria by physical contact, comprising a sterile aqueous solution of about 10- 100 micrograms per milliliter or streptomycin and about 20- 200 micrograms per milliliter of quinacrine hydrochloride.
 4. A composition for killing bacteria by physical contact, comprising a sterile aqueous solution of about 0.01- 1.0 unit of penicillin per milliliter and about 20- 200 micrograms per milliliter of quinacrine hydrochloride.
 5. A composition for killing bacteria by physical contact comprising a sterile aqueous solution of about 0.1- 10 micrograms per milliliter of novobiocin and about 20- 200 micrograms per milliliter of quinacrine hydrochloride.
 6. A composition for killing bacteria by physical contact comprising a sterile aqueous solution of about 0.1-10.0 micrograms per milliliter of erythromycin and about 20-200 micrograms per milliliter of quinacrine hydrochloride.
 7. A composition for killing bacteria by physical contact comprising a sterile aqueous solution of about 0.1- 10.0 micrograms per milliliter of tetracycline and about 20- 200 micrograms per milliliter of quinacrine hydrochloride.
 8. A composition for killing bacteria by physical contact comprising a sterile aqueous solution of about 0.1- 10 micrograms per milliliter of chloramphenicol and about 20- 200 micrograms per milliliter of quinacrine hydrochloride.
 9. A composition for killing bacteria by physical contact comprising a sterile aqueous solution of about 0.1- 100 micrograms per milliliter of sulfathiazole and about 20- 200 micrograms per milliliter of quinacrine hydrochloride. 